Project Summary Dimethyl fumarate (DMF, Tecfidera?) is a first-line treatment for patients with relapsing forms of multiple sclerosis that affects more than one million people in the U.S. DMF is a prodrug that after oral administration undergoes complete presystemic metabolism to its active metabolite, monomethyl fumarate (MMF). The metabolic pathway involved in conversion of DMF to MMF remains uncertain, but our preliminary work shows that human carboxylesterase-1 (CES1), an enzyme highly expressed in the liver, is primarily responsible. Furthermore, as has been shown with other CES1 substrate drugs, DMF hydrolysis to MMF is inhibited by ethanol and it undergoes transesterification with ethanol to form new ethylated metabolites of unknown pharmacology. The overall objective in this application is to determine how ethanol affects DMF metabolism and disposition in vitro and in vivo. The overall hypothesis is that ethanol will decrease MMF active metabolite formation and produce the transesterification metabolite, ethyl methyl fumarate. This hypothesis will be tested by pursuing two specific aims: (1) Identify the DMF-ethanol transesterification product; and (2) determine the effect of ethanol on DMF disposition in a mouse model. In the first aim, human recombinant CES1 will be used to determine the effects of ethanol on DMF hydrolysis in vitro and to identify the transesterification product. For the second aim, a novel plasma carboxylesterase-deficient mouse model will be used to characterize how ethanol affects the in vivo disposition of DMF. This new knowledge will provide important insight into the potential of ethanol to alter the safety and effectiveness of DMF therapy in patients with multiple sclerosis. The impact of ethanol?s effects on DMF therapy are highly relevant as ethanol has been reported to decrease the progression of disability in patients with multiple sclerosis and the prevalence of ethanol consumption in patients with multiple sclerosis is similar to the general population. Thus, the interaction between DMF and ethanol will be a common occurrence in patients on chronic, lifelong DMF therapy, which could alter the effectiveness of DMF in the treatment of their multiple sclerosis.